One of the principal mechanisms by which cellular regulation is effected is through the transduction of extracellular signals across the membrane that in turn modulate biochemical pathways within the cell. Protein phosphorylation represents one course by which intracellular signals are propagated from molecule to molecule resulting finally in a cellular response. These signal transduction cascades are highly regulated and often overlapping as evidenced by the existence of many protein kinases as well as protein phosphatases. It is currently believed that a number of disease states and/or disorders are a result of either aberrant expression or functional mutations in the molecular components of kinase cascades. Consequently, considerable attention has been devoted to the characterization of these proteins.
The cot oncogene (also known as Tpl-2 and est) was first identified in a rearranged form in embryonic SHOK (Syrian hamster osaka kanazawa) cells transformed by transfection with genomic DNA from human thyroid carcinoma cells (Miyoshi et al., Mol. Cell. Biol., 1991, 11, 4088-4096). The gene encoding the normal cellular product of the cot oncogene was later isolated from a Ewing sarcoma cell line and found to reside on the long arm of human chromosome 10 (Chan et al., Oncogene, 1993, 8, 1329-1333).
The cot oncogene protein has been characterized as a serine/threonine kinase of the MAP kinase kinase kinase family of enzymes and, as such, has been shown to participate in several kinase pathways including the MAPK (Kim et al., J. Cell. Biochem., 1998, 71, 286-301), JNK/SAPK (Hagemann et al., Oncogene, 1999, 18, 1391-1400; Troppmair et al., J. Biol. Chem., 1994, 269, 7030-7035) and NF-kappa-B kinases (Belich et al., Nature, 1999, 397, 363-368; Lin et al., Immunity, 1999, 10, 271-280). Ectopic expression of the cot oncogene has been shown to induce the transcription of interleukin-2 in T-cells, suggesting that cot oncogene may also be involved in the CD3/CD28 costimulatory pathway (Ballester et al., J. Immunol., 1997, 159, 1613-1618). More recently, it has been demonstrated that the cot oncogene activates tumor necrosis factor (TNF)-alpha gene expression in a cyclosporin A-resistant manner. In these studies, overexpression of cot oncogene in Jurkat cells (a human T-cell leukemia cell line) promoted TNF-alpha production while a kinase-inactive form of the enzyme did not (Ballester et al., J. Biol. Chem., 1998, 273, 14099-14106).
The cot enzyme is localized to the cytoplasm and exists in two different forms, a long form and a truncated or short form, which are products of alternative initiation mechanisms. These two forms have differing transforming activities and it is rearrangement of the C-terminus that increases cellular transformation (Aoki et al., Oncogene, 1991, 6, 1515-1519; Aoki et al., J. Biol. Chem., 1993, 268, 22723-22732). Expression of cot oncogene is increased upon treatment of cells with interleukin-1, a cytokine involved in inflammation or okadaic acid, a well-known tumor promoter (Chan et al., Oncogene, 1993, 8, 1329-1333). In the rat, it has been demonstrated that the cot oncogene is involved in mouse mammary tumor virus (MMTV) associated transformation of mammary gland cells (Erny et al., Oncogene, 1996, 13, 2015-2020). The pharmacological modulation of cot oncogene activity and/or expression may therefore be an appropriate point of therapeutic intervention in pathological conditions such as inflammation, cancer and disorders of the immune system.
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of cot oncogene and to date, investigative strategies aimed at modulating cot oncogene function have involved the use of antibodies and kinase-inactive mutants. Consequently, there remains a long felt need for agents capable of effectively inhibiting cot oncogene function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of cot oncogene expression.
The present invention provides compositions and methods for modulating cot oncogene expression, including modulation of the alternative short form of the cot oncogene.